CN109790540A - Extract kit used in the method and this method of nucleic acid - Google Patents
Extract kit used in the method and this method of nucleic acid Download PDFInfo
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Abstract
The present invention discloses a kind of method that nucleic acid is extracted from organism sample.This method has: the step of organism sample, anionic surfactant and chaotropic compound are obtained by mixing lysate by (i)；(ii) alkanol, chaotropic compound and silicon dioxide granule that lysate, boiling point are more than 75 DEG C are mixed and makes step of the nucleic acid absorption on silicon dioxide granule；(iii) the step of silicon dioxide granule for being adsorbed with nucleic acid being cleaned using cleaning solution；And (iv) will be adsorbed on silicon dioxide granule using eluent Nucleic Acid Elution the step of, at least step (i) and (ii) more than 75 DEG C at a temperature of carry out.
Description
Technical field
The present invention relates to kits used in the method and this method of extracting nucleic acid.
Background technique
As one of the method for extracting nucleic acid from organism sample, there is BOOM method (such as referenced patent document 1).At this
In method, make nucleic acid absorption on silicon dioxide granule in the presence of chaotropic compound (chaotropic compound), it will
Nucleic acid is separated from organism sample, which is to seize hydrate water from the organisms macromolecule such as nucleic acid and make the life
The higher structure of object molecule becomes unstable substance.
As the method using BOOM method, such as following methods can be enumerated, this method, which has, dissolves organism sample
The step of and in the presence of chaotropic compound and/or alkanol by silica etc. and DNA immobilization the step of, wherein
Implement the immobilization (such as referenced patent document 2) of the nucleic acid within the temperature range of 36~75 DEG C.
Existing technical literature
Patent document
Patent document 1: Japanese Unexamined Patent Publication 2-289596 bulletin
Patent document 2: Japanese Unexamined Patent Application Publication 2008-529509 bulletin
Summary of the invention
Problem to be solved by the invention
In order to extract nucleic acid, carries out destroying the membrane structure of cell, content is made to be discharged into extracellular dissolving step.
As one of dissolving method, the enzyme dissolution method using protease is widely used in commercially available nucleic acid extraction kit.Separately
Outside, in the case where extracting nucleic acid from the cell of gram-positive bacteria or fungi etc. with special cells wall in microorganism,
It needs to be pre-processed furthermore with cell wall clastic enzymes such as lysozyme, yeast lyases.Therefore, according to the kind of biomaterial
Class, corresponding kit or operating instruction are usually different.
In addition, for most of kits using enzyme dissolution methods, relative to the Overall Steps time of nucleic acid extraction,
Dissolving step occupies most of the time.In addition, needing further progress step with centrifugal separation in most cases.Therefore, no
It is suitable for requiring the application of rapidity.
The present invention is in view of such actual situation to complete, and therefore, the purpose of the present invention is to provide can shorten
Kit used in method and this method that extract the nucleic acid desired time from organism sample, extraction nucleic acid.
The method for solving problem
The present invention provides a kind of method that nucleic acid is extracted from organism sample, has: (i) is by organism sample, yin
The step of ionic surfactant and chaotropic compound are obtained by mixing lysate；It (ii) is more than 75 DEG C by lysate, boiling point
Alkanol, chaotropic compound and silicon dioxide granule mixing and make step of the nucleic acid absorption on silicon dioxide granule；(iii) sharp
The step of silicon dioxide granule for being adsorbed with nucleic acid is cleaned with cleaning solution；And (iv) will be adsorbed on two using eluent
The step of Nucleic Acid Elution on silicon oxide particle, at least step (i) and (ii) more than 75 DEG C at a temperature of carry out.
In the above method, preferred steps (iv) more than 75 DEG C at a temperature of carry out.
Boiling point is more than that 75 DEG C of alkanol can be 1- propyl alcohol.
Anionic surfactant is preferably lithium dodecyl sulfate.
Chaotropic compound can be guanidine salt.
In addition, the present invention provides a kind of nucleic acid extraction kit, and it includes: first containing anionic surfactant
Solution is used in dissolution；The second dissolution solution containing chaotropic compound；It and is more than 75 DEG C of alkanol, chaotropic chemical combination containing boiling point
The adsorption liquid of object and silicon dioxide granule.
In kit, boiling point is more than that 75 DEG C of alkanol can be 1- propyl alcohol.
In kit, anionic surfactant is preferably lithium dodecyl sulfate.
In kit, chaotropic compound can be guanidine salt.
Invention effect
Nucleic acid desired time, extraction core are extracted from organism sample according to the present invention it is possible to provide and can shorten
Kit used in the method and this method of acid.
Detailed description of the invention
Fig. 1 (a)~(e) is the figure that the extracted amount of the nucleic acid obtained using Full automatic instrument for extracting nucleic acid is shown respectively.(a) it is
Figure using the result in the case where the sample containing pneumococcus is shown, is (b) to show to use the sample containing bacillus pertussis
In the case where result figure, be (c) figure shown using the result in the case where the sample containing saccharomycete, be (d) to show
Using the type adenovirus of II containing someone sample in the case where result figure, use (e) is shown containing influenza A virus
The figure of result in the case where sample.
Fig. 2 (a), (b) are the figures that the extracted amount of the nucleic acid obtained using Full automatic instrument for extracting nucleic acid is shown respectively.(a) it is
The figure of result in the case where showing using the sample for being added with the sample containing pneumococcus in the sample of Healthy People source,
(b) be show using in the sample of Healthy People source added with the type of II containing someone adenovirus sample sample in the case where knot
The figure of fruit.
Fig. 3 (a), (b) are the figures that the relationship of extracted amount of eluting temperature and nucleic acid is shown respectively.It (a) is that use is shown to contain
There is the figure of the result in the case where the sample of pneumococcus, is (b) the case where showing using sample containing influenza A virus
Under result figure.
Specific embodiment
Hereinafter, present embodiment is described in detail.But the present invention is not limited to embodiment party below
Formula.
The method for extracting nucleic acid in the slave organism sample of present embodiment has (i) for organism sample, anionic
The step of surfactant and chaotropic compound are obtained by mixing lysate.
Lysate is for example by the way that organism sample is added into the miniature tube configured on heat block, contains anionic table
The first dissolution solution of face activating agent and the second dissolution solution containing chaotropic compound are simultaneously mixed, are heated and obtained
It arrives.
By organism sample, the first dissolution solution containing anionic surfactant and chaotropic can be contained
The the second dissolution solution for closing object is mixed simultaneously, by organism sample and can also contain anionic surfactant
First dissolution solution or the second dissolution containing chaotropic compound are obtained by mixing mixing with any one solution in solution
After liquid, then the mixed liquor mixed with another solution.
In this specification, " organism sample " refers to the sample for nucleic acid extraction, specifically, be virus, bacteriophage,
Part or all in bacterium, fungi, the cell of biology, tissue and organ, in addition to the sample that is directly acquired from organism with
It outside, further include the sample obtained in the environment such as water, soil, air.As organism sample, such as blood, pharynx can be enumerated
Swab Nasopharyngeal swabs, is coughed up phlegm, cerebrospinal fluid, urine, excrement, saliva etc..
Nucleic acid can be the naturally occurring nucleic acid such as DNA and RNA.
As anionic surfactant, such as lithium dodecyl sulfate (Lithium Dodecyl can be enumerated
Sulfate, LDS), lauryl sodium sulfate etc..From being not easy from the viewpoint of being precipitated at low temperature, anionic surfactant
Preferably lithium dodecyl sulfate (Lithium Dodecyl Sulfate, LDS).
As chaotropic compound, can enumerate such as guanidine salt.Guanidine salt for example can for (different) guanidine thiocyanate (GuSCN) and
Guanidine hydrochloride.
After step (i), lysate, boiling point are more than 75 DEG C of alkanol, chaotropic compound and titanium dioxide silicon grain by (ii)
Son mixing, makes nucleic acid absorption on silicon dioxide granule.
In order to make nucleic acid absorption on silicon dioxide granule, for example, having above-mentioned dissolution to being arranged on heat block and being added
Addition is more than 75 DEG C of alkanol, the adsorption liquid of chaotropic compound and silicon dioxide granule and progress containing boiling point in the miniature tube of liquid
Mixing, heating.
From the viewpoint of shortening is dissolved to the absorption desired time, the implementation temperature of step (i) and step (ii) are super
Cross 75 DEG C of temperature, preferably 80 DEG C or more, more preferably 85 DEG C or more.Step (i) and the implementation temperature of step (ii) are preferred
For 100 DEG C hereinafter, more preferably 98 DEG C hereinafter, further preferably 95 DEG C or less.The implementation temperature of step (i) and step (ii)
For example, 75~100 DEG C, 75~98 DEG C, 75~95 DEG C, 80~100 DEG C, 80~98 DEG C, 80~95 DEG C, 85~100 DEG C, 85~
98 DEG C or 85~95 DEG C.
Implementation temperature by making step (i) and step (ii) is that can shorten the temperature of liquid temperature more than 75 DEG C of temperature
Degree changes the desired time.Therefore, the absorption desired time for being dissolved to nucleic acid of organism sample can be shortened.Its result
It is the extraction desired time that can also shorten nucleic acid (from the elution desired time for being dissolved to nucleic acid of organism sample).
The boiling point of alkanol used in step (ii) is the temperature more than 75 DEG C, preferably 80 DEG C or more, more preferably 85
DEG C or more.The boiling point of alkanol used in step (ii) be preferably 120 DEG C hereinafter, more preferably 110 DEG C hereinafter, further preferably
It is 100 DEG C or less.The boiling point of alkanol used in step (ii) is, for example, 75~120 DEG C, 75~110 DEG C, 75~100 DEG C, 80
~120 DEG C, 80~110 DEG C, 80~100 DEG C, 85~120 DEG C, 85~110 DEG C or 85~100 DEG C.
Boiling point is more than that 75 DEG C of alkanol is, for example, 1- propyl alcohol, ethyl alcohol, isopropanol etc..
The concrete example of chaotropic compound is identical as the concrete example of chaotropic compound in step (i).
As silicon dioxide granule, it can enumerate by alkyl silica, alumina silicate (also referred to as " zeolite "), there is amino
The compositions such as silica particle.From the viewpoint of being easy to gather, silicon dioxide granule is preferably magnetic silica grain
Son.
After step (ii), (iii) cleans the silicon dioxide granule for being adsorbed with nucleic acid using cleaning solution.
Step (iii) can for example have: addition contains pure and mild anion in the silicon dioxide granule for being adsorbed with nucleic acid
First cleaning solution of type surfactant is simultaneously stirred, and then will be adsorbed with the silicon dioxide granule and the first cleaning solution of nucleic acid
The step of separation；And addition contains polyethylene glycol (Polyethylene Glycol) in silicon dioxide granule after isolation
The second cleaning solution and the step of be stirred, then separate the silicon dioxide granule for being adsorbed with nucleic acid with the second cleaning solution.
Alcohol can be 2- propyl alcohol, 1- propyl alcohol or ethyl alcohol.Alcohol can be their mixture.
Anionic surfactants such as can for lauryl sodium sulfate (Sodium Dodecyl Sulfate,
SDS), or lithium dodecyl sulfate.Anionic surfactant can be their mixture.
The average molecular weight of polyethylene glycol (Polyethylene Glycol) can be 200~10000.As such
Polyethylene glycol (Polyethylene Glycol) can enumerate such as Macrogol 4000 (Polyethylene Glycol
4000,PEG4000).In this specification, the average molecular weight of polyethylene glycol refers in the Japanese Pharmacopoeia revised according to the 16th time
The value of the average molecular weight test measurement of each Macrogol (polyethylene glycol) recorded.
After step (iii), Nucleic Acid Elution that (iv) will be adsorbed on silicon dioxide granule using eluent.
Nucleic acid can use such as following methods and be eluted.That is, firstly, by be adsorbed with nucleic acid silicon dioxide granule and
Eluent is mixed and stirred for, and is then heated.After being again stirring for, silicon dioxide granule is separated with solution, thus, it is possible to will
Nucleic Acid Elution.
From the viewpoint of in a short time efficiently by adsorbed Nucleic Acid Elution, the implementation temperature of step (iv) is super
Cross 75 DEG C of temperature, preferably 80 DEG C or more, more preferably 85 DEG C or more.The implementation temperature of step (iv) be preferably 100 DEG C with
Under, more preferably 98 DEG C hereinafter, further preferably 95 DEG C or less.The implementation temperature of step (iv) is, for example, 75~100 DEG C, 75
~98 DEG C, 75~95 DEG C, 80~100 DEG C, 80~98 DEG C, 80~95 DEG C, 85~100 DEG C, 85~98 DEG C or 85~95 DEG C.
By the way that the implementation temperature of step (iv) to be set as to the temperature more than 75 DEG C in the same manner as the implementation temperature of step (ii)
Degree, can carry out the extraction of nucleic acid (from organism sample in the case where not changing the heating condition of the heaters such as heat block
It is dissolved to the elution of nucleic acid).Therefore, the temperature change desired time in heater with solution can be shortened, it can be further
Shorten the extraction desired time of nucleic acid (from the elution desired time for being dissolved to nucleic acid of organism sample).
Implementation temperature by making step (i), step (ii) and step (iv) be more than 75 DEG C of temperature, can be short
The nucleic acid of high receipts amount is obtained from organism sample in time.In addition, if the implementation temperature of step (i), step (ii) and step (iv)
Degree be more than 75 DEG C of temperature, then can under the conditions of the temperature of equal extent implementation steps (i), step (ii) and step
(iv), as long as therefore single heater, do not need temperature and set different multiple heaters.
Eluent can enumerate such as aqua sterilisa, low salt concn buffer.The buffer of low salt concn is, for example, to contain
There is the buffer of the Tris hydrochloric acid (Tris-HCl) of 10mM.
It can be carried out manually from above-mentioned steps (i) to step (iv), instrument for extracting nucleic acid also can be used and fully automatically carry out.
Each solution required for the method for present embodiment can be packed in advance, in the form of nucleic acid extraction kit
It utilizes.That is, nucleic acid extraction kit includes the first dissolution solution containing anionic surfactant.Anionic surface
Anionic surfactant in the step of concrete example of activating agent is with from the method for extracting nucleic acid in organism sample (i)
Concrete example is identical.
The concentration of anionic surfactant is with the concentration in the mixed liquor of organism sample and the first dissolution solution
Meter is preferably 0.01~10 mass %, more preferably 0.1~5 mass %, further preferably 0.5~3 mass %.
First dissolution solution can further contain Tris hydrochloric acid (Tris-HCl) and ethylenediamine tetra-acetic acid
(Ethylenediaminetetraacetic Acid、EDTA)。
Nucleic acid extraction kit can further include the second dissolution solution containing chaotropic compound.
The concrete example of chaotropic compound with from organism sample extract nucleic acid method the step of (i) in chaotropic chemical combination
The concrete example of object is identical.
The concentration of the chaotropic compound contained in second dissolution solution is with organism sample, the first dissolution solution and
Densimeter in the mixed liquor of two dissolutions solution is preferably 0.01~5M, more preferably 0.1~4.5M, further preferably 1
~4M.
Second dissolution solution further can contain Tris hydrochloric acid (Tris-HCl).
It is more than 75 DEG C of alkanol, chaotropic compound and titanium dioxide containing boiling point that nucleic acid extraction kit, which can be further included,
The adsorption liquid of silicon particle.
Boiling point be more than 75 DEG C of alkanol, the concrete example of chaotropic compound and silicon dioxide granule and preferred embodiment with from biology
The boiling point extracted in the method for nucleic acid in body sample is more than 75 DEG C of alkanol, the concrete example of chaotropic compound and silicon dioxide granule
It is identical with preferred embodiment.
It with the densimeter in the mixed liquor of above-mentioned lysate and adsorption liquid is preferably 1 that boiling point, which is more than the concentration of 75 DEG C of alkanol,
~99 mass %, more preferably 10~90 mass %, further preferably 20~70 mass %.
The concentration of the chaotropic compound contained in adsorption liquid is with the densimeter in the mixed liquor of above-mentioned lysate and adsorption liquid
Preferably 0.01~5M, more preferably 0.1~4M, further preferably 1~4.5M.
Nucleic acid extraction kit can further include the first cleaning from the method for extracting nucleic acid in organism sample
Liquid, the second cleaning solution and eluent.
The nucleic acid extraction kit of present embodiment can be suitable for the application of wide scope.Nucleic acid according to the present embodiment
Extracts kit can utilize same reagent box from blood, throat swab, Nasopharyngeal swabs, cough up phlegm, cerebrospinal fluid, urine, excrement, saliva
Nucleic acid is extracted in the biomaterials of wide scopes such as bacterium, fungi, the virus contained in equal clinical samples.It is furthermore possible to also provide with
General method, which is compared to shorten, extracts kit used in method and this method of the desired time, extraction nucleic acid.
Embodiment
Hereinafter, based on embodiment, more specifically the present invention will be described, but the present invention is not limited to embodiments.
Use bacterium shown in following table 1 and virus.Pneumococcus is acquired from culture plate (S.pneumoniae, to be also referred to as
It for " S.P "), is suspended in physiological saline, the bacterial concentration of McF#1 is adjusted to by turbidimetric analysis turbidimetry, be used as sample (1).
Bacillus pertussis (B.pertussis, being also referred to as " B.P ") is acquired from culture plate, is suspended in physiological saline, is passed through turbidimetric analysis turbidimetry
It is adjusted to the bacterial concentration of McF#1, is used as sample (2).Saccharomycete is acquired from culture plate (S.cerevisiae, to be also referred to as
It for " S.C. "), is suspended in physiological saline, the bacterium solution for being equivalent to OD=6.0 is adjusted to by the turbidimetric analysis turbidimetry under 600nm wavelength
Concentration is used as sample (3).People II type adenovirus is carried out to A549 cell (Human Adenovirus 2, to be also referred to as
" ADV ") infection culture, by the regenerant recycled from culture supernatant be used as sample (4).Flu-A disease is carried out to mdck cell
The infection culture of poison (H3N2 type is also referred to as " FluA "), is used as sample (5) for its culture supernatant.
[table 1]
| Use strain | |
| Pneumococcus (S.pneumoniae, S.P) | ATCC49619 |
| Bacillus pertussis (B.pertussis, B.P) | Clinical separation strain |
| Saccharomycete (S.cerevisiae, S.C) | BY611 |
| People's II type adenovirus (Human Adenovirus 2, ADV) | ATCCVR-846 |
| Influenza A virus (H3N2 type, FluA) | Clinical separation strain |
As nucleic acid extracting reagent, the solution of composition as shown below is prepared.
First dissolution solution: 233.3mM Tris-HCl (pH7.5), 23.3mM EDTA, 4.6 (w/w) %LDS
Second dissolution solution: 100mM Tris-HCl (pH7.5), 4.25M GuSCN
Adsorption liquid: 47.3 (w/w) %1- propyl alcohol, 2.5M GuSCN, silica magnetic particle
First cleaning solution: 41.0 (w/w) %2- propyl alcohol, 1M NaCl, 1.1 (w/w) %SDS
Second cleaning solution: 9.5 (w/w) %PEG4000,1M NaCl
Eluent: 10mM Tris-HCl (pH7.5)
(embodiment 1: utilizing the extraction of the nucleic acid of Full automatic instrument for extracting nucleic acid)
By following step, extracted from S.P, B.P, S.C, ADV, FluA in fully automated manner using instrument for extracting nucleic acid
Nucleic acid.That is, firstly, in 200 μ L of physiological saline add (1) 3 μ L of S.P sample, obtain organism sample A.Similarly in physiology
(2) 2 μ L of B.P sample, (3) 3 μ L of S.C sample, (4) 3 μ L of ADV sample, (5) 1 μ L of FluA sample are added in 200 μ L of salt water respectively,
Obtain organism sample B~E.After organism sample A~E is mixed with the first dissolution with 150 μ L of solution in miniature tube, stirring
7 seconds.Then, it is heated 1 minute on 110 DEG C of heat block.The state that holding is heated with heat block, further addition second is molten
Solution 350 μ L of solution is heated 1 minute on 110 DEG C of heat block after stirring 7 seconds.The state that holding is heated with heat block,
800 μ L of adsorption liquid is further added, is then stirred 7 seconds.Then, it after being heated 1 minute on 110 DEG C of heat block, stirs 7 seconds
Clock further heats 1 minute on 110 DEG C of heat block.The silica magnetic particle for being adsorbed with nucleic acid is separated with solution
Afterwards, it adds 900 μ L of the first cleaning solution and stirs 7 seconds.It will be adsorbed with the silica magnetic particle and solution point of nucleic acid again
From rear, addition 900 μ L of the second cleaning solution is simultaneously stirred 7 seconds.Silica magnetic particle is collected, after discarding supernatant, addition elution
250 μ L of liquid is simultaneously stirred 7 seconds.After standing 2 minutes on 110 DEG C of heat block, stirs 7 seconds, the dioxy of nucleic acid will be adsorbed with
SiClx magnetic particle is separated with solution, obtains nucleic acid extraction liquid I~V.It should be noted that being monitored to liquid temperature, confirmation exists
Liquid temperature is more than 75 DEG C in dissolving step, adsorption step and elution step.
Using the nucleic acid in obtained nucleic acid extraction liquid I~V as template, PCR reaction is carried out, nucleic acid is quantified.
That is, firstly, being implemented under reagent composition as shown below and reaction condition using primer sets shown in following table 2 and probe
PCR.For embodiment 1, carries out 3 same extractions and quantify.By the result of average value and standard error be shown in Fig. 1 (a)~
(e)。
[table 2]
The condition of composition and the PCR reaction of PCR reaction solution
Nucleic acid extraction liquid I or IV
2×Premix Ex TaqTM(Takara) 12.5μL
10 μM of 0.5 μ L of forward primer
10 μM of 0.5 μ L of reverse primer
10 μM of 0.5 μ L of probe
6 μ L of water (Sigma) without deoxyribonuclease, ribalgilase
5 μ L of template
95 DEG C of 10 seconds 1 time circulations of thermal denaturation
Amplification [95 DEG C 5 seconds60 DEG C 20 seconds] 40 circulations
Nucleic acid extraction liquid II
2×Premix Ex TaqTM(Takara) 10μL
10 μM of 1.8 μ L of forward primer
10 μM of 1.8 μ L of reverse primer
10 μM of 1.0 μ L of probe
0.4 μ L of water (Sigma) without deoxyribonuclease, ribalgilase
5 μ L of template
95 DEG C of 15 seconds 1 time circulations of thermal denaturation
Amplification [95 DEG C 3 seconds57 DEG C 30 seconds] 40 circulations
Nucleic acid extraction liquid III
2×Premix Ex TaqTM(Takara) 12.5μL
50 μM of 0.25 μ L of forward primer
50 μM of 0.25 μ L of reverse primer
10 μM of 0.5 μ L of probe
6.5 μ L of water (Sigma) without deoxyribonuclease, ribalgilase
5 μ L of template
95 DEG C of 10 seconds 1 time circulations of thermal denaturation
Amplification [95 DEG C 5 seconds60 DEG C 20 seconds] 40 circulations
Nucleic acid extraction liquid V
2×QuantiTect Probe RT-PCR Master Mix(QIAGEN) 12.5μL
50 μM of 0.3 μ L of forward primer
50 μM of 0.3 μ L of reverse primer
5 μM of 0.5 μ L of probe
QuantiTect RT Mix(QIAGEN) 0.25μL
0.1 μ L of 20IU/ μ L ribonuclease inhibitor
6.05 μ L of water (Sigma) without deoxyribonuclease, ribalgilase
5 μ L of template
50 DEG C of 30 minutes 1 time circulations of reverse transcription
95 DEG C of 15 minutes 1 time circulations of thermal denaturation
Amplification [94 DEG C 15 seconds56 DEG C 75 seconds] 45 circulations
(embodiment 2: utilizing the extraction of the pneumococcus nucleic acid in the Healthy People source sample of Full automatic instrument for extracting nucleic acid)
Sample (1) is blended in the sample of Healthy People source and extracts nucleic acid.Firstly, in 200 μ L of throat swab suspension
(1) 3 μ L of sample is added, organism sample F is obtained.Similarly, sample (1) 3 is added in 200 μ L of Nasopharyngeal swabs suspension respectively
μ L, in 200 μ L of serum add (1) 3 μ L of sample, in 200 μ L of blood (anti-coagulants containing EDTA-2K) addition (1) 3 μ L of sample,
(1) 3 μ L of sample is added in blood (containing sodium citrate anticoagulant) 200 μ L, is added in 200 μ L of blood (agent containing anticoagulant heparin)
(1) 3 μ L of sample, obtains organism sample G~K.Nucleic acid extraction liquid VI~X is obtained similarly to Example 1.
For obtained nucleic acid extraction liquid VI~X, PCR reaction is carried out similarly to Example 1, nucleic acid is determined
Amount.
It should be noted that in the production of organism sample H, using using purified water by the powder serum in Healthy People source
Sample obtained from recovery implements 3 times and extracts and quantitatively, obtain the result of N=3 as Healthy People source sample.Organism examination
In the production of sample F, G, I, J, K, the Healthy People sample of 3 donors is used respectively, is implemented 1 time and is extracted and quantify, as N=3
Result.The result of N=3 is shown in Fig. 2 (a) with average value and standard error.In addition, as control is compared, it is also shown that implement
The result (physiological saline) of extracting solution I in example 1.
(embodiment 3: utilizing the extraction of the adenoviral nucleic acid in the Healthy People source sample of Full automatic instrument for extracting nucleic acid)
Sample (1) is replaced using sample (4), in addition to this, nucleic acid extraction liquid is obtained similarly to Example 2, carries out PCR
Reaction is to quantify.Show the result in Fig. 2 (b).In addition, as control is compared, it is also shown that the result of the extracting solution IV in embodiment 1
(physiological saline).
(embodiment 4: the relationship of eluting temperature and the recovery efficiency of pneumococcus genomic DNA)
Eluting temperature be room temperature (25 DEG C), 50 DEG C, implement in the set environments of 80 DEG C or 110 DEG C, in addition to this, with reality
It applies example 1 and extracting solution is similarly obtained by the organism sample A of sample (1), carry out PCR reaction, nucleic acid is quantified.By result
It is shown in Fig. 3 (a).
(embodiment 5: the relationship of eluting temperature and the recovery efficiency of influenza A genes group RNA)
Eluting temperature be room temperature (25 DEG C), 50 DEG C, implement in the set environments of 80 DEG C or 110 DEG C, in addition to this, with reality
It applies example 1 and extracting solution is similarly obtained by the organism sample A of sample (1), carry out PCR reaction, nucleic acid is quantified.By result
It is shown in Fig. 3 (b).
(reference example 1)
Using QIAamp (registered trademark) DNA Mini Kit (QIAGEN corporation), from the organism containing S.P
Nucleic acid is extracted in sample.That is, replacing centrifugal sediment as sample using sample (1)；And use 10mM Tris-HCl
(pH7.5) buffer solution A E or purified water is replaced to extract nucleic acid according to the recommendation operating instruction of kit in addition to this.It needs
Illustrate, the recommendation operating instruction of kit is QIAamp DNA Mini and Blood Mini Handbook (the 5th edition)
Middle record, Appendix D:Protocol for Bacteria, Isolation of genomic DNA form Gram-
Positive bacteria (Appendix D: bacterium operating instruction-genomic DNA is separated from gram-positive bacterium) and
Protocol:DNA purification from Tissues (DNA operating instruction: is purified from tissue).For using
The nucleic acid that QIAamp (registered trademark) DNA Mini Kit (QIAGEN corporation) extracts, similarly to Example 1 into
Row PCR reaction, quantifies nucleic acid.Show the result in Fig. 1 (a).
(reference example 2)
Using QIAamp (registered trademark) DNA Mini Kit (QIAGEN corporation), from the organism containing B.P
Nucleic acid is extracted in sample.That is, replacing centrifugal sediment as sample using sample (2)；Implement Proteinase K processing in 10 minutes；
And buffer solution A E or purified water are replaced using 10mM Tris-HCl (pH7.5), in addition to this, is grasped according to the recommendation of kit
Nucleic acid is extracted as regulation.It should be noted that the recommendation operating instruction of kit is QIAamp DNA Mini and Blood
Recorded in Mini Handbook (the 5th edition), Appendix D:Protocol for Bacteria, Isolation of
(Appendix D: bacterium operating instruction-is from Bacteria Culture plate culture by genomic DNA from bacterial plate cultures
Genomic DNA is separated in object) and Protocol:DNA purification from Tissues (operating instruction: from tissue
Purify DNA).For the nucleic acid extracted, nucleic acid is quantified in the same manner as reference example 1.Show the result in Fig. 1 (b).
(reference example 3)
Using QIAamp (registered trademark) DNA Mini Kit (QIAGEN corporation), from the organism containing S.C
Nucleic acid is extracted in sample.That is, replacing centrifugal sediment as sample using sample (3) respectively；Implement at 10 minutes Proteinase Ks
Reason；And buffer solution A E or purified water are replaced using 10mM Tris-HCl (pH7.5), in addition to this, according to the recommendation of kit
Operating instruction extracts nucleic acid.It should be noted that the recommendation operating instruction of kit is QIAamp DNA Mini and
Recorded in Blood Mini Handbook (the 5th edition), Appendix E:Protocol for Yeast (annex E: yeast behaviour
Make regulation) and Protocol:DNA purification from Tissues (DNA operating instruction: is purified from tissue).It is right
In the nucleic acid extracted, nucleic acid is quantified in the same manner as reference example 1.Show the result in Fig. 1 (c).
(reference example 4)
Using QIAamp (registered trademark) MinElute virus centrifugal column kit (QIAGEN corporation), from containing ADV
Organism sample in extract nucleic acid.That is, instead of the blood plasma or serum recorded in the recommendation operating instruction of kit, using in life
Sample obtained from (4) 3 μ L of sample is added in reason 200 μ L of salt water to mention according to the recommendation operating instruction of kit in addition to this
Take nucleic acid.It should be noted that the recommendation operating instruction of kit is QIAamp (registered trademark) MinElute Virus Spin
Recorded in Handbook (version in 2010), Protocol:Purification of Viral Nucleic Acids from
Plasma or Serum (operating instruction: the purified virus nucleic acid from blood plasma or serum).
For using QIAamp (registered trademark) MinElute virus centrifugal column kit (QIAGEN corporation) to extract
Nucleic acid, similarly to Example 1 carry out PCR reaction to quantify.Show the result in Fig. 1 (d).
For Examples 1 to 3 and reference example 1~4, will extract the desired time is shown in following Table 3.
[table 3]
Sequence table
<110>Eiken Chemical (Eiken Chemical CO., LTD)
<120>kit (METHOD FOR EXTRACTING NUCLEIC used in the method and this method of nucleic acid is extracted
ACIDS AND KIT FOR THE SAME)
<130> FP17-0614-00
<150> JP 2016-193987
<151> 2016-09-30
<160> 15
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213>artificial sequence
<220>
<223>pneumococcus forward primer (S.P forward primer)
<400> 1
acgcaatcta gcagatgaag c 21
<210> 2
<211> 21
<212> DNA
<213>artificial sequence
<220>
<223>pneumococcus reverse primer (S.P reverse primer)
<400> 2
tgtttggttg gttattcgtg c 21
<210> 3
<211> 25
<212> DNA
<213>artificial sequence
<220>
<223>pneumococcus probe (S.P probe)
<400> 3
tttgccgaaa acgcttgata caggg 25
<210> 4
<211> 19
<212> DNA
<213>artificial sequence
<220>
<223>bacillus pertussis forward primer (B.P forward primer)
<400> 4
atcaagcacc gctttaccc 19
<210> 5
<211> 21
<212> DNA
<213>artificial sequence
<220>
<223>bacillus pertussis reverse primer (B.P reverse primer)
<400> 5
ttgggagttc tggtaggtgt g 21
<210> 6
<211> 22
<212> DNA
<213>artificial sequence
<220>
<223>bacillus pertussis probe (B.P probe)
<400> 6
aatggcaagg ccgaacgctt ca 22
<210> 7
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>saccharomycete forward primer (S.C forward primer)
<400> 7
ggactctgga catgcaagat 20
<210> 8
<211> 21
<212> DNA
<213>artificial sequence
<220>
<223>saccharomycete reverse primer (S.C reverse primer)
<400> 8
atacccttct taacacctgg c 21
<210> 9
<211> 30
<212> DNA
<213>artificial sequence
<220>
<223>saccharomycete probe (S.C probe)
<400> 9
cccttcagag cgttttctct aaattgatac 30
<210> 10
<211> 23
<212> DNA
<213>artificial sequence
<220>
<223>adenovirus forward primer (ADV forward primer)
<400> 10
gccaccgtgg ggtttctaaa ctt 23
<210> 11
<211> 25
<212> DNA
<213>artificial sequence
<220>
<223>adenovirus reverse primer (ADV reverse primer)
<400> 11
gccgcagtgg tcttacatgc acatc 25
<210> 12
<211> 29
<212> DNA
<213>artificial sequence
<220>
<223>adenovirus probe (ADV probe)
<400> 12
tgcaccaggc cccggctcag gtactccga 29
<210> 13
<211> 29
<212> DNA
<213>artificial sequence
<220>
<223>influenza A virus forward primer (FluA forward primer)
<220>
<221> misc_feature
<222> (3)..(3)
<223> m is a or c
<400> 13
ccmaggtcga aacgtaygtt ctctctatc 29
<210> 14
<211> 32
<212> DNA
<213>artificial sequence
<220>
<223>influenza A virus reverse primer (FluA reverse primer)
<400> 14
tgacagraty ggtcttgtct ttagccaytc ca 32
<210> 15
<211> 22
<212> DNA
<213>artificial sequence
<220>
<223>influenza A probe (FluA probe)
<400> 15
atytcggctt tgagggggcc tg 22
Claims (9)
1. a kind of method that nucleic acid is extracted from organism sample,
It has: the organism sample, anionic surfactant and chaotropic compound are obtained by mixing lysate by (i)
The step of；
(ii) alkanol, chaotropic compound and silicon dioxide granule that the lysate, boiling point are more than 75 DEG C are mixed and is made described
Step of the nucleic acid absorption on the silicon dioxide granule；
(iii) the step of silicon dioxide granule for being adsorbed with the nucleic acid being cleaned using cleaning solution；And
(iv) the step of using eluent by the Nucleic Acid Elution on the silicon dioxide granule is adsorbed on,
At least step (i) and (ii) more than 75 DEG C at a temperature of carry out.
2. the method for claim 1, wherein the step (iv) more than 75 DEG C at a temperature of carry out.
3. method according to claim 1 or 2, wherein the boiling point is more than that 75 DEG C of alkanol is 1- propyl alcohol.
4. method according to any one of claims 1 to 3, wherein the anionic surfactant is dodecyl
Lithium sulfate.
5. method as described in any one of claims 1 to 4, wherein the chaotropic compound is guanidine salt.
6. a kind of nucleic acid extraction kit, it includes:
The first dissolution solution containing anionic surfactant,
The second dissolution solution containing chaotropic compound and
It is more than 75 DEG C of alkanol, the adsorption liquid of chaotropic compound and silicon dioxide granule containing boiling point.
7. kit as claimed in claim 6, wherein the boiling point is more than that 75 DEG C of alkanol is 1- propyl alcohol.
8. kit as claimed in claims 6 or 7, wherein the anionic surfactant is lithium dodecyl sulfate.
9. the kit as described in any one of claim 6~8, wherein the chaotropic compound is guanidine salt.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2016-193987 | 2016-09-30 | ||
| JP2016193987 | 2016-09-30 | ||
| PCT/JP2017/033741 WO2018061877A1 (en) | 2016-09-30 | 2017-09-19 | Nucleic acid extraction method and kit using same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109790540A true CN109790540A (en) | 2019-05-21 |
Family
ID=61759667
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201780058975.1A Pending CN109790540A (en) | 2016-09-30 | 2017-09-19 | Extract kit used in the method and this method of nucleic acid |
Country Status (5)
| Country | Link |
|---|---|
| US (1) | US20200032241A1 (en) |
| EP (1) | EP3521447B1 (en) |
| JP (1) | JPWO2018061877A1 (en) |
| CN (1) | CN109790540A (en) |
| WO (1) | WO2018061877A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114958830A (en) * | 2022-06-27 | 2022-08-30 | 爱科睿特生物医疗科技(南京)有限公司 | Kit for simultaneously extracting pathogen DNA and RNA and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1572796A (en) * | 2003-05-19 | 2005-02-02 | 株式会社日立高新技术 | Method and apparatus for recovering nucleic acids |
| CN101115833A (en) * | 2005-02-11 | 2008-01-30 | 恰根有限公司 | Method for isolating nucleic acids, the nucleic acids being immobilised on a matrix at an increased temperature |
| CN102834518A (en) * | 2010-01-08 | 2012-12-19 | 霍夫曼-拉罗奇有限公司 | Improved recovery of nucleic acids from magnetic glass particles |
| JP2014030364A (en) * | 2012-08-01 | 2014-02-20 | Seiko Epson Corp | Dna extraction method |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4196185B2 (en) * | 2002-07-29 | 2008-12-17 | Ｊｓｒ株式会社 | Nucleic acid separation method |
| US20080268451A1 (en) * | 2007-03-30 | 2008-10-30 | Bruce Seligmann | Measurement of an insoluble analyte in a sample |
| EP2345719A1 (en) * | 2010-01-18 | 2011-07-20 | Qiagen GmbH | Method for isolating small RNA |
-
2017
- 2017-09-19 CN CN201780058975.1A patent/CN109790540A/en active Pending
- 2017-09-19 US US16/337,279 patent/US20200032241A1/en not_active Abandoned
- 2017-09-19 JP JP2018542427A patent/JPWO2018061877A1/en active Pending
- 2017-09-19 EP EP17855832.6A patent/EP3521447B1/en active Active
- 2017-09-19 WO PCT/JP2017/033741 patent/WO2018061877A1/en unknown
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1572796A (en) * | 2003-05-19 | 2005-02-02 | 株式会社日立高新技术 | Method and apparatus for recovering nucleic acids |
| CN101115833A (en) * | 2005-02-11 | 2008-01-30 | 恰根有限公司 | Method for isolating nucleic acids, the nucleic acids being immobilised on a matrix at an increased temperature |
| CN102834518A (en) * | 2010-01-08 | 2012-12-19 | 霍夫曼-拉罗奇有限公司 | Improved recovery of nucleic acids from magnetic glass particles |
| JP2014030364A (en) * | 2012-08-01 | 2014-02-20 | Seiko Epson Corp | Dna extraction method |
Also Published As
| Publication number | Publication date |
|---|---|
| EP3521447A1 (en) | 2019-08-07 |
| WO2018061877A1 (en) | 2018-04-05 |
| EP3521447A4 (en) | 2020-05-13 |
| US20200032241A1 (en) | 2020-01-30 |
| JPWO2018061877A1 (en) | 2019-06-24 |
| EP3521447B1 (en) | 2021-08-11 |
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